Regulation of systemic immune responses utilizing cytokines and antigens

ABSTRACT

A method of altering the specific, systemic immune response of an individual to a target antigen by the co-administration of a cytokine an adhesion or accessory molecule and the target antigen. The target antigen may be a tumor cell, a tumor cell antigen, an infectious agent or other foreign antigen, or other antigens to which an enhanced systemic immune response is desirable. Alternatively, the antigen may be a non-foreign antigen when a suppression of a systemic immune response is desired. The resulting systemic immune response is specific for the target antigen.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 08/265,554,filed Jun. 23, 1994, now U.S. Pat. No. 5,637,483, which is afile-wrapper-continuation of U.S. application Ser. No. 07/956,621, filedOct. 5, 1992, abandoned, which is a continuation-in-part of U.S.application Ser. No. 07/771,194, filed Oct. 4, 1991, abandoned, thedisclosures of which are incorporated herein by reference.

I. FIELD OF THE INVENTION

The present invention in all of its associated aspects relates to amethod of altering an individual's immune response to a target antigenor antigens. More particularly, the present invention is concerned withco-administering a target antigen and at least one cytokine, in such amanner as to either increase or decrease the individual's immuneresponse. Possible antigens include tumor cells, tumor cell antigens,infectious agents, foreign antigens, or non-foreign components. Theinvention also relates to methods and combinations for co-administeringthe antigens and the cytokines.

II. BACKGROUND OF THE INVENTION

The immune system plays a critical role in the pathogenesis of a widerange of important diseases and conditions, including infection,autoimmunity, allograft rejection and neoplasia. The shortcomings of theimmune system in these disorders can be broadly considered as either thefailure to develop a sufficiently potent response to a deleterioustarget or the inappropriate generation of a destructive response againsta desirable target. Standard medical treatments for these diseases,including chemotherapy, surgery and radiation therapy, have clearlimitations with regard to both efficiency and toxicity. Whileprevention of the disease or condition would be ideal, these approachestypically have met with little success. New strategies based on specificmanipulation of the immune response are greatly needed.

III. DESCRIPTION OF THE PRIOR ART

For the convenience of the reader, the references referred to in thetext are listed numerically in parentheses. These numbers correspond tothe numerical references listed in the appended bibliography. By thesereferences, they are hereby expressly incorporated by reference herein.

The use of autologous cancer cells as vaccines to augment anti-tumorimmunity has been explored throughout this century (1). However, theimmunogenicity of cancer cells is generally too weak to elicit apronounced immune reaction sufficient to overcome the disease. Patientresponses to these "raw" vaccines have generally been only partial andrelatively shortlived. Strategies to improve the efficacy of suchvaccinations, including the use of non-specific immunostimulants such asBCG and Corynebacterium parvum, have resulted in little improvement.

One approach has been to increase the immunogenicity of tumor cells bytreating the cells in different ways. For example, U.S. Pat. No.4,931,275 describes using as a vaccine either cells treated withpressure or cholesteryl hemisuccinate, or plasma membranes or membraneproteins from these cells. These methods attempt to enhance the exposureof surface antigens to render the antigens more immunogenic.

Another approach focuses on the interaction of cytokines and the immunesystem. Cytokines and combinations of cytokines have been shown to playan important role in the stimulation of the immune system. For example,U.S. Pat. No. 5,098,702, describes using combinations of TNF, IL-2 andIFN-β in synergistically effective amounts to combat existing tumors.U.S. Pat. No. 5,078,996 describes the activation of macrophagenonspecific tumoricidal activity by injecting recombinant GM-CSF totreat patients with tumors.

A further expansion of this approach involves the use of geneticallymodified tumor cells to evaluate the effects of cytokines ontumorigenicity. Since the doses of cytokines necessary to effect tumordevelopment are often systemically toxic, direct treatment of patientsis frequently not feasible (see, for example, (2) and (3)). Therefore,other methods of cytokine delivery are being developed. Studies showthat localized high concentrations of certain cytokines, delivered bygenetically modified cells, lead to tumor regression (4, 5). Suchstudies show that the transduction of murine tumor cells with variouscytokine genes can lead to the rejection of the genetically modifiedcells by syngeneic hosts. For example, growth of malignant mouseneuroblastoma cells injected into mice was strongly suppressed when thecells constitutively expressed γ-IFN at high levels (4,6). Injection ofmammary adenocarcinoma tumor cells expressing IL-4, mixed with a varietyof nontransfected tumor cells, inhibited or prevented tumor formation ofall types (7). Similar results are seen for IL-2 (8,9), TNF-α (2,10,11),G-CSF (12), JE (13), and IL-7 (14).

Yet another approach involves the use of genetically modified tumorcells as vaccines. Injection of tumor cells expressing IL-2 not onlysuppresses tumor formation initially but confers a short-lived systemicimmunity as well, thus allowing the mice to reject a subsequentchallenge of tumor cells (8). For example, mice were able to rejecttumor cells injected two weeks after the vaccine but not at four weeks.This immunity is tumor specific in that other tumors grew normally wheninjected two weeks after the initial vaccine injection (8). Similarresults are seen with TNF-α, where animals that experience initial tumorrejection were challenged two weeks later and did not develop new tumorsfor at least 40 days (2). A somewhat longer systemic immunity is seenwith IFN-γ, with mice vaccinated with IFN-γ producing tumor cellssuccessfully rejecting unmodified tumor cells injected 6 weeks later.

The efficacy of these vaccines to stimulate the immune system to attacka previously growing tumor, which is the more desirable trait of thevaccine, is not as clear. For IL-4, IFN-γ, and IL-2, injection ofcytokine-producing tumor cells did not affect the growth of non-modifiedtumor cells at a different site on the animal. It is undocumentedwhether the non-modified tumor cells were already established tumors, orcells that were simultaneously injected with the modified cytokineproducing tumor cells (7, 4, 9 and 8, respectively). In a single recentcase, tumor cells engineered to secrete IL-4 successfully mediatedrejection of established renal cancer cells (15).

While these studies may suggest the potential use of gene transfer as ameans of augmenting anti-tumor immunity, there are many problems withthese systems. First of all, treating already existing cancers is one ofthe primary goals of the research. While the vaccine aspect of thesemethods is very important, particularly in precancerous patients, forexample, or in cases where certain individuals acquire defined cancers(such as AIDS patients), the ability to systemically treat existingcancers is particularly appealing. Another problem is the relativelyshort period of efficacy for the vaccine; generally these vaccines havebeen effective only for tumor challenges at less than 2 to 6 weeks aftervaccination.

Also, the mechanism by which engineered cytokine expression promotessuch immunity is currently unclear. To date, for instance, none of thegene transfer studies have compared the efficacy of such live vaccinesto the immune response induced simply by the vaccination of hosts withnon-transduced cells, either inactivated by γ-irradiation or othermeans, or by non-transduced cells that are surgically remove afterimplantation. This would appear to be quite important, since a largebody of literature indicates that often such vaccination schemes alonecan stimulate potent anti-tumor immunity (16, 17, 18, 19).

IV. SUMMARY OF THE INVENTION

The present invention is based on the determination that tumor cellsexpressing certain cytokines and combinations of cytokines can conferlong term specific systemic immunity to individuals receiving injectionsof such cells. From that finding, the present invention provides for theregulation, either in a stimulatory or suppressive way, of anindividual's immune response to a useful antigen.

The method of the present invention is useful for preventative purposesas well as therapeutic applications. That is, it is useful to protect anindividual against development or progression of a tumor, bacterial orviral infection such as AIDS, rejection of transplanted tissue, orautoimmune condition. The present invention also will find utility inthe reversal or suppression of an existing tumor, condition or disease,such as established tumors, bacterial or viral infections such as AIDS,transplanted tissue rejection, or autoimmune responses. In addition, thepresent invention finds utility in the treatment of chronic and lifethreatening infections, such as the secondary infections associated withAIDS, as well as other bacterial, fungal, viral, parasitic and protozoalinfections. A particular advantage of the present invention is that thecytokines may be selected to optimize effects in the individual and thusmaximize the desired result.

As one aspect of the present invention, there is disclosed a method forregulating the immune response of an individual to a target antigen. Theregulation is achieved by co-administering to the individual the targetantigen and at least one cytokine, adhesion or accessory molecules orcombinations thereof, in such a manner that there is a systemic immuneresponse. The antigen and the cytokine, adhesion or accessory moleculesor combinations thereof, are co-administered in a therapeuticallyeffective amount, which results in the systemic immune response.

Another aspect of the present invention utilizes cells which express thetarget antigen and at least two cytokines, either naturally or as aresult of genetic engineering, that can be administered to an individualwhose immune response to the target antigen is to be regulated. Forexample, a tumor cell of the type against which an enhanced immuneresponse is desired can be engineered to express the cytokines to beadministered. The resulting genetically engineered tumor cell is used asa vaccine, to protect against future tumor development or as a deliveryvehicle to result in the reversal of previously existing tumors.

Specific aspects of the present invention utilize tumor cells expressingthe two cytokines GM-CSF and IL-2, and utilizes melanoma cells as thetumor cells to be modified.

Another aspect of the present invention relates to the use of modifiedtumor cells, expressing cytokines, which are irradiated or renderedproliferation incompetent prior to administration to an individual.These irradiated, modified cells are administered in therapeuticallyeffective amounts. Administration of these cells results in theregulation, either as a stimulatory manner or a suppressive manner, ofthe individual's systemic immune response. Of particular utility aretumor cells expressing GM-CSF, and specifically melanoma cellsexpressing GM-CSF. Also of use in the present invention are irradiatedtumor cells expressing GM-CSF IL-4, IL-6, CD2 and ICAM.

Human clinical studies are also described that involve autologousprimary and/or secondary melanoma or renal cell carcinoma cells whichhave been transduced to express the cytokine GM-CSF which have beenirradiated to an extent that the cells are no longer capable ofsustained cell division, and injected into the original donor.

In other aspects, the invention relates to the use of modified tumorcells expressing cytokines for the reversal or suppression ofpre-existing tumors. The tumor cells may express two cytokines, forexample IL-2 and GM-CSF, or three cytokines, for example IL-2, GM-CSF,and TNF-α. The third cytokine may also be IL-4, CD2 or ICAM. The tumorcells may also be irradiated or rendered proliferation incompetent byother means prior to administration.

In another aspect, the present invention relates to the use ofretroviral vectors to genetically engineer the cytokine-expressing tumorcells. The invention may utilize a single infection of a tumor cell by aretroviral vector encoding a cytokine, or it may utilize multipleinfections by retroviral vectors encoding different cytokines.

A variety of retroviral vectors may be used. The MFG, α-SGC, pLJ, andpEm vectors abandoned, (and derivatives thereof, e.g. MFG-S) are morefully disclosed in U.S. Ser. No. 07/786,015, filed Oct. 31, 1991abandoned (PCT/US91/08121, filed Oct. 31, 1991), incorporated herein byreference, and will find particular utility in the present invention.

V. DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically represents the recombinant retroviral vectorsuseful in the present invention. FIG. 1A shows a detailed representationof the MFG vector. FIG. 1B shows the pLJ vector. FIG. 1C shows the pEmvector. FIG. 1D shows the A-SGC vector.

FIG. 2:

FIG. 2A graphically represents the ability of B16 melanoma cellsexpressing IL-2 to protect against subsequent challenge with wild-typeB16 melanoma cells at various times after vaccination

FIG. 2B graphically represents the ability of B16 melanoma cellsexpressing IL-2 and a second cytokine (as indicated) after vaccinationto protect against challenge with wild-type B16 melanoma cells. Thesymbol ◯ represents the animals that succumbed to tumor challenge andthe symbol  represents the animals protected from tumor challenge.

FIG. 2C graphically represents the ability of B16 melanoma cellsexpressing IL-2 and GM-CSF and a third cytokine (as indicated) toprotect against subsequent challenge with wild-type B16 melanoma cells.

FIG. 3:

FIG. 3A graphically represents the ability of irradiated B16 melanomacells expressing GM-CSF to protect against challenge with wild-type B16melanoma cells. The symbol ◯ represents the animals that succumbed totumor challenge and the symbol  represents the animals protected fromtumor challenge.

FIG. 3B graphically represents the ability of irradiated B16 melanomacells expressing GM-CSF to protect against challenge with wild-type B16melanoma cells either at varying doses or when mixed with non-transducedB16 melanoma cells. The symbol ◯ represents the animals that succumbedto tumor challenge and the symbol  represents the animals protectedfrom tumor challenge.

FIG. 3C graphically represents the ability of irradiated B16 melanomacells expressing GM-CSF to increase the immune response afterinoculation with live non-transduced B16 melanoma cells (pre-establishedtumor) as indicated. The symbol ◯ represents the animals that succumbedto tumor challenge and the symbol  represents the animals protectedfrom tumor challenge.

FIG. 4 graphically represents the ability of irradiated B16 melanomacells expressing a cytokine (as indicated) to protect against subsequentchallenge with wild-type B16 melanoma cells. The symbol ◯ represents theanimals that succumbed to tumor challenge and the symbol  representsthe animals protected from tumor challenge.

FIG. 5 pictorially represents the histological analysis of the site ofB16 melanoma vaccination. FIG. 5A shows the site of irradiated GM-CSFtransduced tumor vaccination. FIG. 5B shows the site of irradiatednon-transduced tumor vaccination site. FIG. 5C shows the draining lymphnode from the irradiated GM-CSF transduced tumor vaccination. FIG. 5Dshows the draining lymph node from the irradiated non-transduced tumorvaccination. FIG. 5E shows the challenge site of a mouse vaccinated withirradiated GM-CSF transduced tumor cells. FIG. 5F shows the challengesite of a mouse vaccinated with irradiated non-transduced tumor cells.FIG. 5G shows the challenge site in a naive mouse.

FIG. 6:

FIG. 6A graphically represents the contribution of lymphocyte subsets tosystemic immunity gene-rated by irradiated GM-CSF transduced B16melanoma cells. The symbol ◯ represents the animals that succumbed totumor challenge and the symbol  represents the animals protected fromtumor challenge.

FIG. 6B graphically represents the CD8 blockable CTL activity,determined by a 4 hour ⁵¹ Cr release assay on gamma-interferon treatedB16 target cells at various effector:target ratios. Fourteen days aftervaccination with either irradiated GM-CSF transduced or non-transducedB16 cells, splenocytes were harvested and stimulated in vitro for 5 dayswith gamma-interferon treated B16 cells.

FIG. 7 graphically represents the immunogenicity of irradiated,non-transduced murine tumor cell lines used in previous cytokinetransfection studies. The symbol ◯ represents the animals that succumbedto tumor challenge and the symbol  represents the animals protectedfrom tumor challenge.

FIG. 8 graphically represents the ability of GM-CSF to enhance thesystemic immunity of irradiated tumor cells of the types indicatedrelative to non-transduced irradiated tumor cells. The symbol ◯represents the animals that succumbed to tumor challenge and the symbol represents the animals protected from tumor challenge.

FIG. 9 graphically represents the effect of B16 melanoma cellsexpressing IL-2, GM-CSF and a third cytokine, as indicated, on apre-existing tumor. The symbol ◯ represents the animals that succumbedto tumor challenge and the symbol  represents the animals protectedfrom tumor challenge.

FIG. 10 shows a time course of the average percentage of eosinophils inthe test subjects during the 64 day period in which the transducedmelanoma cells were injected into the patients. The arrows indicate thatthe injections were made at days 1, 22, and 43. The circles representthe average data of the low dose patients and the triangles indicate theaverage data of the high dose patients.

VI. DETAILED DESCRIPTION OF THE INVENTION

6.1. Definitions

By the term "regulating the immune response" or grammatical equivalents,herein is meant any alteration in any cell type involved in the immuneresponse. The definition is meant to include an increase or decrease inthe number of cells, an increase or decrease in the activity of thecells, or any other changes which can occur within the immune system.The cells may be, but are not limited to, T lymphocytes, B lymphocytes,natural killer (NK) cells, macrophages, eosinophils, mast cells,dendritic cells, or neutrophils. The definition encompasses both astimulation or enhancement of the immune system to develop asufficiently potent response to a deleterious target, as well as asuppression of the immune system to avoid a destructive response to adesirable target. In the case of stimulation of the immune system, thedefinition includes future protection against subsequent tumorchallenge.

By the term "cytokine" or grammatical equivalents, herein is meant thegeneral class of hormones of the cells of the immune system, bothlymphokines and monokines, and others. The definition is meant toinclude, but is not limited to, those hormones that act locally and donot circulate in the blood, and which, when used in accord with thepresent invention, will result in an alteration of an individual'simmune response. The cytokine can be, but is not limited to, IL-l(α orβ), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,GM-CSF, M-CSF, G-CSF, LIF, LT, TGF-β, γ-IFN (or α or β-IFN), TNF-α,BCGF, CD2, or ICAM. Descriptions of the aforementioned cytokines as wellas other applicable immunomodulatory agents may be found in "Cytokinesand Cytokine Receptors", A. S. Hamblin, 1993, (D. Male, ed., OxfordUniversity Press, New York, N.Y.), or the "Guidebook to Cytokines andTheir Receptors", 1995, N. A. Nicola, ed. (Oxford University Press, NewYork, N.Y.) herein incorporated by reference.

Where therapeutic use in humans is contemplated, the cytokines orhormones will preferably be substantially similar to the human form ofthe protein or have been derived from human sequences (i.e., of humanorigin).

Additionally, cytokines of other mammals with substantial homology tothe human forms of IL-2, GM-CSF, TNF-α, and others, will be useful inthe invention when demonstrated to exhibit similar activity on theimmune system. Similarly, proteins that are substantially analogous toany particular cytokine, but have relatively minor changes of proteinsequence, will also find use in the present invention. It is well knownthat some small alterations in protein sequence may be possible withoutdisturbing the functional abilities of the protein molecule, and thusproteins can be made that function as cytokines in the present inventionbut differ slightly from currently known sequences. Thus, proteins thatare substantially similar to any particular cytokine, but haverelatively minor changes of protein sequence, will also fine use in thepresent invention.

Finally, the use of either the singular or plural form of the word"cytokine" in this application is not determinative and should not limitinterpretation of the present invention and claims. In addition to thecytokines, adhesion or accessory molecules or combinations thereof, maybe employed alone or in combination with the cytokines.

By the term "antigen from a tumor cell" or grammatical equivalentsthereof, herein is meant any protein, carbohydrate or other componentcapable of eliciting an immune response. The definition is meant toinclude, but is not limited to, using the whole tumor cell with all ofits associated antigens as an antigen, as well as any componentseparated from the body of the cell, such as plasma membranes, proteinspurified from the cell surface or membrane, or unique carbohydratemoieties associated with the cell surface. The definition also includesthose antigens from the surface of the cell which require specialtreatment of the cells to access.

By the term "systemic immune response" or grammatical equivalentsherein, is meant an immune response which is not localized, but affectsthe individual as a whole, thus allowing specific subsequent responsesto the same stimulus.

By the term "co-administering" or grammatical equivalents herein, ismeant a process whereby the target antigen and the selected cytokine orcytokines are encountered by the individual's immune system atessentially the same time. The components need not be administered bymeans of the same vehicle. If they are administered in two separatevehicles, they must be administered sufficiently closely, both in timeand by route of administration, that they are encountered essentiallysimultaneously by the individual's immune system to achieve the desiredspecificity.

By the term "reversal of an established tumor" or grammaticalequivalents herein is meant the suppression, regression, partial orcomplete disappearance of a preexisting tumor. The definition is meantto include any diminution in the size, potency, growth rate, appearanceor feel of a pre-existing tumor.

One of ordinary skill will appreciate that, from a medicalpractitioner's or patient's perspective, virtually any alleviation orprevention of an undesirable symptom (e.g., symptoms related to disease,sensitivity to environmental or factors, normal aging, and the like)would be desirable. Thus, for the purposes of this Application, theterms "treatment", "therapeutic use", or "medicinal use" used hereinshall refer to any and all uses of the claimed compositions which remedya disease state or symptoms, or otherwise prevent, hinder, retard, orreverse the progression of disease or other undesirable symptoms in anyway whatsoever.

By the term "therapeutically effective amount" or grammaticalequivalents herein refers to an amount of the preparation that issufficient to regulate, either by stimulation or suppression, thesystemic immune response of an individual. This amount may be differentfor different individuals, different tumor types and different cytokinepreparations. An appropriate dosage of transduced cells, or derivativesthereof, may be determined by any of several well establishedmethodologies. For instance, animal studies are commonly used todetermine the maximal tolerable dose, or MTD, of bioactive agent perkilogram weight. In general, at least one of the animal species testedis mammalian. Those skilled in the art regularly extrapolate doses forefficacy and avoiding toxicity to other species, including human. Beforehuman studies of efficacy are undertaken, Phase I clinical studies innormal subjects help establish safe doses. Alternatively, initialtoxicity studies may involve individuals that are at the terminal stagesof the disease progression.

By the term "rejection" or grammatical equivalents herein is meant asystemic immune response that does not allow the establishment of newtumor growth.

By the term "challenge" or grammatical equivalents herein is meant asubsequent introduction of tumor cells to an individual. Thus a"challenge dose 5 days post vaccination" means that on the fifth dayafter vaccination with tumor cells expressing cytokines or irradiatedtumor cells, or both, a dose of unmodified tumor cells was administered."Challenge tumor" means the tumor resulting from such challenge.

By the term "days to sacrifice" or grammatical equivalents herein, ismeant that period of time before mice were sacrificed. Generally, micewere sacrificed when challenge tumors reached 2-3 centimeters in longestdiameter, or if severe ulceration or bleeding developed.

By the term "irradiated cells" or "inactivated cells" or grammaticalequivalents herein is meant cells inactivated by rendering themproliferation incompetent by irradiation. This treatment results incells which are unable to undergo mitosis, but still retain thecapability to express proteins such as cytokines. Typically a minimumdose of about 3500 rads is sufficient, although doses up to about 30,000rads are acceptable. It is understood that irradiation is but one way toinactivate the cells, and that other methods of inactivation whichresult in cells incapable of cell division but that retain the abilityto express cytokines are included in the present invention (i.e.,treatment with mitomycin C and conceptually analogous agents).

By the term "individual" or grammatical equivalents herein is meant anyone individual mammal.

The present invention relates to a method of regulating the immuneresponse of an individual to target antigens by administering a mixtureof the target antigen or antigens and a cytokine, or cytokines, in sucha manner that the immune system of the individual is either stimulatedor suppressed. In either case, the present invention provides a means ofcontrolling the activity of the cytokines with the result that theyprovide an unusual protective or therapeutic effect.

In one embodiment of the present invention, the target antigen and oneor more cytokines, adhesion or accessory molecules or combinationsthereof, are administered to an individual in a chemical compositionwhich provides for transfer or release of the cytokines, adhesion oraccessory molecules or combinations thereof, in direct proximity or incombination with the target antigen. The two components need not beadministered in the same vehicle. If they are administered in twoseparate vehicles, they must be administered sufficiently closely, bothin time and by route of administration, that they are encounteredessentially simultaneously by the individual's immune system. That is,the cytokines, adhesion or accessory molecules or combinations thereof,can be co-administered with the target antigen in any manner whichprovides transfer or delivery of the cytokine in the context of thetarget antigen in relation to which the immune response is to beregulated. For example, this can be accomplished by using slow orsustained release delivery systems, or direct injection. As a result ofthis co-administration, the nonspecific cytokine has the specific effectof amplifying or altering the specific immune response to the targetantigen. The emphasis is on local interaction of the cytokine and thetarget antigen to mimic the physiological occurrence of simultaneouspresentation of cytokine and antigen, to maximize efficacy and minimizetoxicity.

In a preferred embodiment, cells which express the target antigen, suchas tumor cells, are themselves genetically engineered to express thecytokines to be administered. The resulting modified tumor cellspresents to the host immune system not only the antigen against which animmune response is sought, but also the cytokines, adhesion or accessorymolecules or combinations thereof, whose expression determines the typeand extent of the resulting immune response. Alternatively, cells thatnaturally express the target antigen and the cytokines can be used. Theresulting cells, which express both the target antigen and thecytokines, can be used as a vaccine, to protect against future tumordevelopment or as a delivery vehicle to result in the reversal ofpreviously existing tumors. Conversely, a target antigen against which adecreased immune response is sought can be used in conjunction with theappropriate cytokine mixture.

In one embodiment, the individual's systemic immune response isincreased or enhanced beyond that which would occur in the absence ofthe co-administered molecules. The systemic response is sufficient forthe individual to mount an immune response against the target antigen,such as the tumor cells, and may cause the tumor cell to be rejected bythe individual.

In another embodiment, the individual's systemic immune response isreduced or suppressed, either partially or completely, to such an extentthat the target antigen, such as antigens on transplanted cells ortissue, or cells of the individual incorrectly recognized as foreign, isnot rejected by the individual, or is rejected to a lesser extent thanwould occur if the present method were not used.

In one embodiment of the present invention, the target antigen can be atumor cell, a tumor cell antigen, an infectious agent or other foreignagent against which an enhanced immune response is desired. For example,any antigen implicated in the progression of a chronic and lifethreatening infection, such as the AIDS virus or a secondary infectionassociated with AIDS, or other bacterial, fungal, viral, parasitic andprotozoal antigens, may be utilized in the present invention.

In a preferred embodiment, the target antigen is a tumor cell. Onepreferred embodiment utilizes melanoma cells, and another embodimentutilizes carcinoma cells. However, other tumor cells such as, but notlimited to, breast cancer cells, leukemia cells, cancerous polyps orpreneoplastic lesions, or cells engineered to express an oncogene (e.g.ras, p53) may also be used. The modified cells may be live or irradiatedcells, or cells inactivated in other ways. Furthermore, the tumor cellsused as the target antigen and expressing cytokines can be either anunselected population of cells or specific clones of modified cells.

In a preferred embodiment, tumor cells are modified to express thecytokines IL-2 and GM-CSF. This combination is particularly desirablesince it results in the long term systemic immune protection againstsubsequent challenge with wild type tumor cells.

In one embodiment of the present invention, tumor cells expressing thetarget antigen and the cytokines are irradiated before administration toan individual. The resulting nonviable tumor cells may be used toregulate the immune response of the individual. The administration ofsuch irradiated cytokine producing tumor cells has been shown to beuseful in inhibiting the establishment of tumors of the same type,presumably through a vaccine-like mechanism. This procedure hasparticular importance since the introduction of live tumor-producingcancer cells to an individual is undesirable. This has additionalsignificance since primary tumor explants likely contain non-neoplasticelements as well, irradiation of the tumor samples prior to vaccinationwill also prevent the possibility of autonomous growth of non-neoplasticcells induced by autocrine synthesis of their own growth factors.

In a preferred embodiment, the irradiated tumor cells used to regulatethe immune response of an individual are capable of expressing GM-CSF,IL-4, IL-6, CD2 or ICAM, or a combination thereof.

In the most preferred embodiment, the irradiated tumor cells used toregulate the immune response of an individual are capable of expressingGM-CSF.

In one embodiment, the administration of the combination of targetantigen and cytokines can be used to reverse established tumors. Thismay be accomplished by taking tumor cells from an individual with apre-existing tumor and modifying these tumor cells to express cytokines.Upon subsequent re-introduction of the modified cells into theindividual, a systemic immune response capable of reversing thepre-existing tumor is produced. Alternatively, tumor cells of the typeof the pre-existing cancer may be obtained from other sources. Theidentification of the cytokines to be expressed will depend on the typeof tumor, and other factors.

In a preferred embodiment of the present invention, the tumor cellsexpress IL-2 and GM-CSF, and a third cytokine from the group TNF-α,IL-4, CD2 and ICAM.

In the most preferred embodiment, the tumor cells express IL-2, GM-CSFand/or TNF-α.

In another embodiment, the modified tumor cells used to reverseestablished tumors are irradiated prior to administration to theindividual.

In a preferred embodiment, the irradiated tumor cells used to reverse anestablished tumor are capable of expressing GM-CSF.

In one embodiment of the present invention, a vector such as theretroviral MFG vector herein described may be used to genetically alterthe vaccinating cells. The MFG vector has particular utility since itsuse makes it feasible, for the first time, to rapidly screen a largenumber of potential immunomodulators for their effects on the generationof systemic immunity and to assess the activity of complex combinationsof molecules. The MFG vector's combination of high titer and high geneexpression obviates the need for selection of transduced cells among thebulk target population. This minimizes the time required for culturingprimary tumor cells prior to vaccination, and maximizing the antigenicheterogeneity represented in the vaccinating inoculum.

Other retroviral vectors will find use in the present invention. Forexample, pLJ, pEm, and α-SGC may be used. pLJ, previously described in(20), is capable of expressing two types of genes: the gene of interestand a dominant selectable marker gene, such as the neo gene. Thestructure of pLJ is represented in FIG. 1B. pEm is a simple vector wherethe gene of interest replaces the gag, pol and env coding sequences ofthe wild-type virus. The gene of interest is the only gene expressed.The structure of pEm is represented in FIG. 1C. The vector α-SGCutilizes transcriptional promoter sequences from the α-globin gene toregulate expression of the gene of interest. The structure of α-SGC isrepresented in FIG. 1D.

On Oct. 3, 1991, Applicants have deposited with the American TypeCulture Collection, Rockville, Md. USA (ATCC) the plasmid MFG with thefactor VIII insertion, described herein ATCC accession no. 68726,plasmid MFG with the tPA insertion, given ATCC accession no. 68727, theplasmid α-SGC, described herein, with the factor VIII insertion, givenATCC accession no. 68728, and plasmid α-SGC with the tPA insertion,given ATCC accession no. 68729. On Oct. 9, 1991, Applicants havedeposited with the ATCC the plasmid MFG, described herein, given ATCCaccession no. 68754, and plasmid α-SGC, described herein and given ATCCaccession no. 68755. These deposits were made under the provisions ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the purposes of patent procedure and the Regulationsthereunder (Budapest Treaty). This assures maintenance of a viableculture for 30 years from the date of deposit. The organisms will bemade available by the ATCC under the terms of the Budapest Treaty, andsubject to an agreement between Applicants and ATCC which assuresunrestricted availability upon issuance of the pertinent U.S. patent.Availability of the deposited strains is not be construed as a licenseto practice the invention in contravention of the rights granted underthe authority of any government in accordance with its patent laws.

One of ordinary skill will realize that the above vectors may be subtlyaltered in a manner such that the operative features vis-a-vistransduction efficacy are substantially maintained. As such, the presentinvention also contemplates the use of any and all operative(transfection competent) derivatives of the above retroviral constructsto deliver genes encoding immunomodulatory agents to cells. For example,the vector MFG-S comprises three point mutations in the MFG vector whichtheoretically improve the safety of the vector (although there is noevidence that MFG is unsafe) while retaining substantially identicaltransfection efficiency. Specifically, MFG-S has an A to T change atnucleotide 1256, a C to T mutation at nucleotide 1478, and a T to an Aat nucleotide 1273.

It follows from the results herein that a variety of hormones, antigens,and cytokines will find use in the present invention. For example,cytokines, antigens, or hormones of other mammals with substantialhomology to the human forms of the cytokines, antigens, and hormones,will be useful in the invention when demonstrated to exhibit similaractivity on the immune system.

Thus, the method of the present invention can include treatment withantigens, hormones, and cytokines, adhesion or accessory molecules orcombinations thereof, combined with other systemic therapy such aschemotherapy, radiation treatments and other biological responsemodifiers.

The method of the present invention can also be used to regulate anindividual's systemic immune response to a variety of antigens. In oneembodiment, the present invention may be used to treat chronic and lifethreatening conditions, such as infection with the AIDS virus, as wellas the secondary infections associated with AIDS. Other embodimentsembrace the treatment of other bacterial, fungal, viral, parasitic, andprotozoal infections described in "Principles and Practice of InfectiousDisease", Mandell et al., eds. 1990, Churchill Livingstone, Inc., NewYork, N.Y.) herein incorporated by reference.

In a preferred embodiment, treatment and prevention of HIV-relatedconditions is achieved. For example, in the case of the humanimmunodeficiency virus (HIV), Simian immunodeficiency virus (SIV) thereare at least six known target antigens (21, 22, 23) against which animmune response can be enhanced using the present invention. Anappropriate host cell expressing the HIV target antigen can be modifiedto express one or more cytokines and administered to an uninfectedindividual, to serve as a vaccine and elicit an enhanced immune responseto confer the ability to resist subsequent infection by the AIDS virus.

Alternatively, the antigen and the cytokines may be administered withouta host cell. Furthermore, co-administration of the target antigen andcytokines can be given to an HIV positive individual, in order to limitthe existing infection or reverse it.

Having described the particular methods employed in the presentinvention for the regulation of systemic immune responses to targetantigens, and detailing how these methods may be utilized, and showingthe successful regulation of systemic immune responses, the presentdisclosure is sufficient to enable one skilled in the art to use thisknowledge to produce the end results by equivalent means using generallyavailable techniques.

The following examples serve to more fully describe the manner of makingand using the above-described invention, as well as to set forth thebest modes contemplated for carrying out various aspects of theinvention. It is understood that these examples in no way serve to limitthe true scope of this invention, but rather are presented forillustrative purposes.

VII. EXAMPLES 7.1. Generation of Recombinant Retroviral Genomes EncodingCytokines

Construction of the retroviral vectors employs standard ligation andrestriction techniques which are well understood in the art. A varietyof retroviral vectors containing a gene or genes encoding a cytokine ofinterest were used.

MFG. This vector is described in co-pending U.S. Ser. No. 07/607,252entitled "Genetic Modification of Endothelial Cells", filed Oct. 31,1990, abandoned U.S. Ser. No. 07/786,015; abandoned "Retroviral VectorsUseful in Gene Therapy", filed Oct. 31, 1991, and PCT/US91/08121, filedOct. 31, 1991, the teachings of which are incorporated herein byreference. They are also described below with particular reference tothe incorporation and expression of genes encoding cytokines.Furthermore, several MFG vectors have been deposited with the TCC asdescribed above.

The MFG vector is similar to the pEm vector, described below anddepicted in FIG. 1, but contains 1038 base pairs of the gag sequence forMoMuLV, to increase the encapsidation of recombinant genomes in thepackaging cells lines, and 350 base pairs derived from MOV-9 whichcontains the splice acceptor sequence and transcriptional start. An 18base pair oligonucleotide containing NcoI and BamHI sites directlyfollows the MOV-9 sequence and allows for the convenient insertion ofgenes with compatible sites. In each case, the coding region of the genewas introduced into the backbone of the MFG vector at the NcoI site andBamHI site. In each case, the ATG initiator methionine codon wassubcloned in frame into the Nco I site and little, if any, sequencebeyond the stop codon was included, in order to avoid destabilizing theproduct and introducing cryptic sites. As a result, the ATG of theinsert was present in the vector at the site at which the wild-typevirus ATG occurs. Thus the splice was essentially the same as occurs inMoloney Murine Leukemia virus and the virus worked very well. The MoMuLVLTR controls transcription and the resulting mRNA contains the authentic5' untranslated region of the native gag transcript followed directly bythe open reading frame of the inserted gene. In this vector, Moloneymurine leukemia virus (Mo-MuLV) long terminal repeat sequences were usedto generate both a full length viral RNA (for encapsidation into virusparticles), and a subgenomic mRNA (analogous to the Mo-MuLV env mRNA)which is responsible for the expression of inserted sequences. Thevector retained both sequences in the viral gag region shown to improvethe encapsidation of viral RNA (24) and the normal 5' and 3' splicesites necessary for the generation of the env mRNA. All oligonucleotidejunctions were sequenced using the dideoxy termination method (25) andT7 DNA polymerase (Sequenase 2). The structure of MFG is represented inFIG. 1A. As can be seen, the virus in marker-free in that it does notcomprise a dominant selectable marker (although one may optionally beinserted), and, given the high levels of transfection efficiency andexpression inherent in the structure of the vector, transduction withMFG derivatives generally does not involve or require a lengthyselection step.

MFG vectors containing genes for the following proteins wereconstructed: murine IL-2, GM-CSF, IL-4, IL-5, γ-IFN, IL-6, ICAM, CD2,TNF-α, and IL-1-RA (interleukin-1-receptor antagonist). In addition,human sequences encoding TNF-α, GM-CSF and IL-2 were constructed (SeeTable 1). It is also possible to make MFG vectors containing a geneencoding one or more of the following: VCAM, ELAM, macrophageinflammatory protein, heat shock proteins (e.g. hsp60), M-CSF, G-CSF,IL-1, IL-3, IL-7, IL-10, TGF-β, B7, MIP-2, MIP-1 α and MIP-1 β.

Precise cDNA sequences subcloned into MFG were as follows: murine IL-2(26) base pairs 49-564; murine IL-4 (27) base paris 56-479; murine IL-5(28) base pairs 44-462; murine GM-CSF (29) base pairs 70-561; murineICAM-1 (30) base pairs 30-1657; murine CD2 (31) base pairs 48-1079;murine IL-1 receptor antagonist (32) base pairs 16-563; human TNF-60(33) base pairs 86-788.

7.2. Cytokine Assays

Cytokines secreted by the infected, unselected B16 populations wereassayed 48 hours after plating 1×10⁶ cells in 10 cm dishes containing 10mls. of medium. IL-1-RA secretion was measured from infected, unselected3T3 cells 24 hours after plating 5×10⁶ cells in a 10 cm dish containing10 mls of medium. Cytokines were assayed as follows: murine IL-2 usingCTLL cells (34) and ELISA (Collaborative Biomedical); murine IL-4 usingCT4R cells (35) and ELISA (Endogen); murine IL-5 using an ELISA(Endogen); murine IL-6 using T1165 cells (41) and ELISA (Endogen);murine GM-CSF using FDCP-1 cells (37) and ELISA (Endogen); murine γ-IFNusing vesicular stomatitis viral inhibition (38) and ELISA (Genzyme);human TNF-α using L929 cells (39) and ELISA (R&D Systems); murineIL-1-RA using ¹²⁵ I-IL1β binding inhibition (40).

Expression of murine ICAM-1 and CD2 in B16 target cells was determinedwith standard procedures (41) on an EPICS-C FACS analyzer (Coulter)using antibodies YN1/1.47 (42) and RM2/1 respectively.

7.3. Production of B16 Melanoma Cells Containing Cytokine-encodingSequences

The above vector constructs were introduced by standard methods into thepackaging cell lines known as Psi CRIP and Psi CRE (43). These celllines have been shown to be useful to isolate clones that stably producehigh titres of recombinant retroviruses with amphotropic and ecotropichost ranges, respectively. Psi CRIP packaging lines with amphotropichost range were generated by both transfection and electroporation withonly small differences in efficiency. Calcium phosphate DNAcoprecipitations were performed (44) using 20 μg of vector and 1 μg ofpSV2NEO (45). Electroporations were performed after linearizing 40 μg ofvector and 8 μg of pSV2NEO using the Gene Pulser electroporator(Bio-Rad). Conditions were 190 V and 960 μF. Producers were placed intoselection in G418 (GIBCO) at 1 mg/ml 36 hours after introduction of theDNA. Both clones and populations of producers were generated.

Viral titres were determined by Southern blot analysis (46) followinginfection (47) of B16 or 3T3 cells in medium containing 8 μg polybreneper ml. Ten μg of infected target cell DNA as well as control DNA spikedwith appropriate copy number standards were digested with Nhe I (an LTRcutter), resolved by electrophoresis in 1% agarose gels, and analyzed bythe method of Southern using standard procedures (48). Blots were probedwith the appropriate sequences which had been labeled to high specificactivity with ³² P!dCTP by the random primer method (49). Probes usedwere all full-length cDNAs except for murine IL-2 which was theSacI/RsaI fragment (base pairs 221-564). A titre of one copy per cell isapproximately equivalent to 1×10⁶ retroviral particles per cell.

During the course of generating these products, we observed thatcross-infection of packaging lines (CRE to CRIP or CRIP to CRE) with MFGvectors sometimes led to the mobilization of packaging function asdetermined by a sensitive his D mobilization assay (43). Suchcross-infection should thus be avoided. No mobilization of packagingfunction with producers generated by direct transfection orelectroporation has been observed.

Viral titres and expression levels are shown in the Table below.

                  TABLE 1    ______________________________________    MFG Vector Constructs                     Titre    Construct        (Ampho)     Expression    ______________________________________    MFG mu IL-2      1.0 copy    5000 U/ml    MFG mu IL-4      0.25 copy   15 ng/ml    MFG mu IL-5      2.0 copy    250 ng/ml    MFG mu IL-6      0.5 copy    400 ng/ml    MFG mu GM-CSF    2.0 copy    300 ng/ml    MFG mu γ-IFN                     0.1 copy    20 ng/ml    MFG mu ICAM      0.5 copy    +FACS    MFG mu CD2       0.5 copy    +FACS    MFG mu ILIRA     1.0 copy    30 ng/ml    MFG hu IL-2      1.0 copy    100 ng/ml    MFG hu GM-CSF    1.0 copy    500 ng/ml    MFG hu TNF       0.5 copy    400 ng/ml    ______________________________________

The B16 melanoma tumor model (50) was chosen for initial studies. Humanmelanoma has been shown to be sensitive to a variety of immunotherapies(51, 52). To examine whether any of the gene products listed in Table 1influenced the growth of B16 cells in vitro or in vivo, cells wereexposed to viral supernatants and the transduced cells werecharacterized for their efficiency of infection and secretion of geneproduct. Table 1 shows the approximate efficiency of infection with eachvirus for the B16 melanoma cell line (a titer of one copy per cellcorresponds to a titer of approximately 10⁶ infectious particles) andthe corresponding level of secreted gene product.

With the exception of the MFG γ-IFN construct, which transmitted atapproximately 0.1 copies/cell, most of the viruses were capable oftransducing a majority of tumor cells. γ-IFN secreting cells grew moreslowly than non-infected cells and adopted a flattened morphologyrelative to their wild type counterparts. In contrast, none of the othertransduced cells displayed any altered in vitro growth characteristics.Both populations and specific clones of virus producing cells were usedin these studies.

7.4. Vaccinations

Tumor cells were trypsinized, washed once in medium containing serum,and then twice in Hanks Balanced Saline Solution (GIBCO) prior toinjection. Trypan blue resistant cells were suspended to the appropriateconcentrations and injected in a volume of 0.5 cc HBSS. Vaccinationswere administered subcutaneously in the abdomen and tumor challengeswere injected in the dorsal midline of the back after anesthetizing themice with Metaphane (Pitman-Moore). Mice were examined at 2-3 dayintervals and the time to development of palpable tumor recorded.Animals were sacrificed when tumors reached 2-3 cm in diameter or ifsevere ulceration or bleeding developed. For antibody depletionexperiments, mice were vaccinated in the right hind leg (volume 0.1 cc)and challenged in the left hind leg (volume 0.1 cc). For establishmentof pulmonary metastases, cells were injected in the tail vein in avolume of 0.2 cc. Animals used were 6-12 week C57B1/6 females (JacksonLabs) for B16, Lewis Lung, and WP-4 experiments, and 6-12 week Balb/cfemales (Jackson Labs) for CT-26, RENCA, and CMS-5 studies.

When irradiated vaccines were employed, tumor cells (after suspension inHBSS) received 3500 rads from a Cesium-137 source discharging 124rads/min.

7.5. Histology

Tissues for histologic examination were fixed in 10% neutral bufferedformalin, processed to paraffin embedment, and stained with hematoxylinand eosin. Tissues for immunoperoxidase staining were snap frozen inliquid N₂, frozen sections were prepared, fixed in acetone for 10minutes, and stored at -20° C. Frozen sections were stained with primaryantibodies, followed by anti-IgG antibodies (species specific), and thenthe antigen-antibody complexes visualized with avidin-biotin-peroxidasecomplexes (Vectastain, Vector Labs) and diaminobenzidine. Primaryantibodies used were 500A2 (hamster anti-CD3), GK 1.5 (rat anti-CD4),and GK2.43 (rat anti-CD8).

7.6. Vaccination Studies with Live Transduced Tumor Cells

To directly assess the effect of the cytokine upon the tumorigenicity ofB16 cells, either unmodified (wild type) B16 melanoma cells or thetransduced modified cells were inoculated subcutaneously into C57B1/6mice, their syngeneic host, and the mice were examined every few daysfor tumor formation. Typically, a vaccinating dose was 5×10⁵ infectedB16 cells.

Surprisingly, only tumor cells secreting IL-2 did not form tumors. Allof the other nine cytokines tested resulted in tumor formation. Modestdelays in tumor formation were associated with expression of IL-4, IL-6,γ-INF, and TNF-α. Several cytokines produced distinctive systemicsyndromes, presumably as a consequence of the progressively increasingnumber of cells. GM-CSF transduced cells induced a fatal toxicitymanifested by profound leukocytosis (polymorphonuclear leukocytes,monocytes, and eosinophils), hepatosplenomegaly, and pulmonaryhemorrhage, IL-5 expressing cells showed a striking peripheraleosinophilia and splenomegaly. IL-6 expressing cells causedhepatosplenomegaly and death. TNF-α expressing cells induced wasting,shivering, and death.

7.7. Systemic Antitumor Immunity Generated by Cytokine-Transduced TumorCells

The rejection of IL-2 transduced cells made it possible to examine theirpotential to generate systemic immunity. Mice were first inoculated withIL-2 expressing B16 melanoma cells, and subsequently challenged over thecourse of one month, with unmodified B16 cells. Typically, both theinitial and challenge dose were 5×10⁵ live cells, cells which had beenharvested from culture dishes and then extensively washed. Cells wereinjected subcutaneously in a volume of about 0.5 cc (in Hanks BufferedSaline (HBS)). Groups of 5 mice were then challenged at a site differentfrom that at which the vaccine was administered. A time course analysiswas carried out by challenging groups of 5 animals every 3-4 days,beginning 3 days after the vaccine was administered and continuing tochallenge the animals until a month after the vaccination.

The results demonstrated that IL-2 possessed a minimal ability to inducesystemic protection, as measured by the ability of the unmodified tumorchallenge to be rejected. In a typical experiment, most mice succumbedto the challenge tumor by 40 days after its inoculation. This is indirect contrast to the results of Fearon et al. (8), since all animalssuccumbed to this challenge, regardless of the time of challenge, withonly an occasional delay in tumor formation, FIG. 2A. Assessment of theeffects of the infected B16 cells was done through ascertaining whennatural death or humane killing of the mice occurred.

7.8. Systemic Antitumor Immunity Generated by MultipleCytokine-Transduced Cells

As a result of the demonstration that tumor cells transduced with IL-2alone may be effectively rejected, combinations of cytokines weretested. For this analysis, populations of B16 cells expressing both IL-2and a second gene product were generated through the superinfection ofIL-2 transduced cells. Animals were vaccinated with the doubly infectedcells and then challenged with non-transduced cells after 7 to 14 days.

Typically, both the initial and challenge doses were 5×10⁵ live cells,cells which had been harvested from culture dishes and then extensivelywashed. Cells were injected subcutaneously in a volume of roughly 0.5 ccof HBS.

B16 melanoma cells were modified to express IL-2 plus one of thefollowing: GM-CSF, IL-4, γ-IFN, ICAM, CD2, ILL-RA, IL-6, or TNF-α.

The results shown in FIG. 2B, indicate that cells expressing both IL-2and GM-CSF generate potent systemic immunity, with a majority of themice surviving tumor challenge long term. For example, mice oftensurvived as long as 120 days, at which time the mice were sacrificed. Intotal, 50 or 60 mice have been vaccinated with this combination, and theresult show that mice were protected from challenge doses ranging from10⁵ unmodified cells (essentially 70% of the animals were protected) to5×10⁶ unmodified cells (which resulted in lesser, but still significantprotection). This result is particularly surprising in light of the factthat tumor cells infected with GM-CSF alone grew progressively in thehost, and, in fact, the host succumbed to the toxicity of GM-CSFsecreted systemically. A small degree of protection was observed withseveral other combinations. TNF-α and IL-4 had a very slight effect incombination with IL-2. IL-6 also showed slight activity when used incombination with IL-2. Under the conditions used, T-IFN, ICAM, CD2 andIL1 receptor antagonists were inactive. Assessment of the effects of theinfected B16 cells was done through ascertaining when natural death orhumane killing of the mice occurred.

In a further set of experiments, the ability of a third cytokine (inaddition to IL-2 and GM-CSF) to affect the ability of that combinationto protect against wild type challenge was also assessed. Thus, B16melanoma cells expressing IL-2, GM-CSF plus either γ-IFN, IL-4, or both,were constructed.

Preliminary results suggest that the addition of IL-4 to IL-2 withGM-CSF improves the efficacy of this approach. See FIG. 2C. Perhaps moreintriguing is that the addition of γ-IFN to IL-2 with GM-CSF compromisesthe ability of the ladder combination to function as an effectivevaccine.

These data suggests that the various combinations of cytokines can bothenhance as well as attenuate the magnitude of the immune response.

7.9. Vaccination Studies With Irradiated Cells 7.9.1. Immunogenicity ofNon-Transduced, Irradiated Cells

The fact that cells expressing both IL-2 and GM-CSF, but not IL-2 alone,conferred systemic protection upon vaccinated hosts, and that cellssecreting GM-CSF alone actually grew progressively, suggested thepossibility that the IL-2 might be functioning primarily to mediaterejection of the vaccinating cells.

Thus, an important control was to assess the vaccination potential ofnon-transduced irradiated cells, since many previous studies have shownthat irradiated tumor cells can possess significant vaccinationactivity. (16, 17, 18, 19). Irradiation and vaccination were done asoutlined above. The data in FIG. 3C indicates that non-transducedirradiated B16 cells elicited only minimal effects upon the growth ofchallenge cells at the vaccine and challenge doses tested.

However, a number of mouse tumor cell lines previously used to identifythe anti-tumor activity of specific cytokines are inherentlyimmunogenic, as revealed by studies involving vaccination withirradiated, non-transduced cells. Furthermore, irradiated cells aloneare able to confer systemic immunity at levels comparable to thoseinduced by live transduced cells. For these experiments, several tumormodels which had been used previously by other investigators to identifycytokines possessing activity in tumor rejection or tumor challengeassays were used. These tumors included: (i) CT26, a colon carcinomaderived cell line (53), used in studies which identified the activity ofIL-2 (8); (ii) CMS-5, a fibrosarcoma derived cell line (54), used toidentify the activity of IL-2 (9) and γ-IFN (4); (iii) RENCA, a renalcell carcinoma derived cell line (55), used to identify the activity ofIL-4 (15); and (iv) WP-4, a fibrosarcoma derived cell line (2), used toidentify the activity of TNF-α (2). For these initial studies, thevaccine and challenge doses were comparable to those used previously inthe cytokine gene transfer studies. The results of tumor challengeassays in which mice vaccinated with irradiated cells were challengedwith tumor cells 7-14 days later are shown in FIG. 7.

Surprisingly, in each case, the irradiated cells possessed potentvaccination activity, comparable to that reported previously with livecells expressing the various cytokines tested above. In contrast, asshown earlier (FIG. 3C), irradiated B16 cells induced little if anysystemic immunity. This has particular significance since it suggeststhat previous work may be misleading. Many of the tumor cells used inprevious studies could have been inherently immunogenic, but thelethality of the live tumor cells masks such characteristics.

The expression of a cytokine may merely mediate the death of the livetumor cells, thus allowing the natural immunogenicity to be expressed.In contrast, the systemic immune responses of the present invention aredue to a real interaction of the immune system and the simultaneouspresentation of cytokine and antigen.

7.9.2. Vaccination With Irradiated, Transduced B16 Cells

As shown above, irradiation of B16 cells expressing both IL-2 and GM-CSFdid not abrogate either their secretion of cytokines in vitro or theirvaccination activity in vivo (data not shown). Based on this result,irradiated B16 cells expressing GM-CSF alone were used to vaccinatemice, and subsequently challenged 7 to 14 days later. Cells wereirradiated using 3500 rads. Typically the irradiated cells were dosed at5×10⁵, with subsequent challenge with the same dose of live cells. Asshown in FIG. 3A, such a vaccination led to potent anti-tumor immunity,with most of the mice surviving their tumor challenge. When non-radiatedcells were used as a vaccine, 1 out of 19 animals was ultimatelyprotected; when irradiated cells were used, 16 out of 20 mice wereprotected. No mice demonstrated the toxicity observed in mice injectedwith live GM-CSF expressing cells and we were unable to detectcirculating GM-CSF in the sera of vaccinated mice using an ELISAsensitive to 10 pg/ml. The systemic immunity was also shown to be longlasting, in that most of the mice vaccinated with irradiated cells whichexpressed GM-CSF and subsequently challenged with non-transduced cellstwo months after vaccination remained tumor free (data not shown).

The systemic immunity was also specific, in that GM-CSF expressing B16cells did not protect mice from a challenge by Lewis Lung carcinomacells, (56), another tumor of C57B1/6 origin, and GM-CSF expressingLewis Lung carcinoma cells did not protect mice from a challenge ofnon-transduced B16 cells (data not shown).

The efficiency of the irradiated transduced cells as a vaccine wasapparent over a wide range of GM-CSF concentrations in that infectedcells could be admixed with a one hundred fold excess of non-transducedcells, with little compromise in systemic immunity, FIG. 3B. This resultmay reflect the relatively high levels of GM-CSF expression afforded bythe MFG vector. In contrast, protection was highly sensitive to thetotal inoculum of vaccinating cells, as activity was severelycompromised with the use of ten fold fewer vaccinating cells. FIG. 3B.

In addition to conferring potent protection against challenge withnon-transduced cells, irradiated B16 cells expressing GM-CSF were alsomore capable of mediating the rejection of pre-established tumors thanwere irradiated cells alone, FIG. 3C. Similar results were also obtainedin studies in which established metastases were generated through theintravenous injection of non-transduced cells (data not shown).

Surprisingly, the localized destruction of vaccinating cells does notalways lead to systemic immunity, as evidenced by the B16 results. Whilelive IL-2 expressing B16 cells were rejected by the syngeneic host, thisvaccination did not generate protection against subsequent challenge ofnon-transduced B16 cells. Similarly, vaccination with non-transducedirradiated B16 cells also failed to induce systemic protection. Thegeneration of systemic immunity in non- or poorly immunogenic tumormodels may thus require qualitatively different mechanisms than thoseresponsible for inducing this immunity in more immunogenic tumor models.

Consistent with this hypothesis is that a screen of many gene productsfor anti-tumor activity in the B16 model, including all of the moleculesidentified in other systems as able to augment such immunity, showedthat a previously unidentified cytokine, GM-CSF, is quite potent.

Demonstration of the advantage of GM-CSF transduction even in tumormodels that are somewhat immunogenic further suggests the relativepotency of GM-CSF expression in comparison to other means of revealingtumor immunogenicity.

Finally, to determine whether irradiated cells expressing other geneproducts might also confer systemic immunity upon vaccinated hosts, asurvey of each of the cell populations expressing different geneproducts (after irradiation) for vaccination activity was done. B16cells expressing IL-1-RA, IL-2, IL-4, IL-5, IL-6, GM-CSF, γ-IFN, TNF,ICAM and CD2 were constructed as described previously. Vaccination andchallenge doses and methods were done as described previously.

The results, shown in FIG. 4, show that B16 cells expressing GM-CSFprior to irradiation appeared to be the most potent, with IL-4 and IL-Gmodified cells showing slightly reduced activity.

7.9.3. Vaccination with Irradiated, GM-CSF Expressing Cells in OtherMurine Tumor Models

Because the significant vaccination activity of the irradiated cells ofthe other murine tumor models alone precluded examination of theactivity of GM-CSF expressing cells, either the challenge or vaccinedose of a number of the tumors examined above were manipulated, in thehopes of establishing conditions where the relative efficiency ofnon-transduced irradiated and irradiated cells expressing GM-CSF couldbe evaluated. These conditions were the same as the ones employedrelating to FIG. 3B. These conditions made possible the comparison ofnon-transduced irradiated and GM-CSF expressing irradiated cells shownin FIG. 8. While the relative efficacy of GM-CSF expressing cells wassomewhat variable from line to line, GM-CSF expressing cells in allcases were more efficacious than irradiated cells alone in elicitingsystemic immunity.

As outlined above, included in this analysis was a study of the LewisLung carcinoma cell line (56), a tumor not previously employed incytokine gene transfer studies. While a representative experimentillustrating the efficacy of GM-CSF transduction is shown, the precisedose which demonstrates this effect has been somewhat variable.

However, the results show that Lewis Lung carcinoma cells which expressGM-CSF prior to irradiation show a specific systemic immunity, in thatmice were protected from a subsequent challenge with unmodified LewisLung carcinoma cells but not from challenge with unmodified B16 melanomacells (data not shown).

The WP-4 cell line was not included in this experiment, since even largechallenge doses were eventually rejected after vaccination withnon-transduced cells (data not shown).

7.10. Reversal of Pre-Existing Tumors Using Cytokine-Transduced TumorCells

The ability of B16 melanoma cells expressing mixtures of cytokines toaffect growth of a pre-existing tumor was tested. All experiments usedmice subcutaneously injected with unmodified B16 cells on day 1. By day7, tumors were microscopically established. On day 7, B16 melanoma cellsexpressing various cytokines were injected at a different subcutaneoussite. Typically, both the initial and challenge doses were 5×10⁵ livecells, cells which had been harvested from culture dishes and thenwashed extensively. Cells were injected subcutaneously in a volume ofroughly 0.5 cc of HBS.

B16 melanoma cells were modified to express the following combinationsof cytokines: IL-2 and GM-CSF; IL-2, GM-CSF and TNF-α; IL-2, GM-CSF andIL-4; IL-2, GM-CSF and IL-6; IL-2, GM-CSF and ICAM; IL-2, GM-CSF andCD2; and IL-2, GM-CSF and IL-1-RA. The results of these studies areshown in FIG. 9.

The experiments showed that if mice with pre-existing tumors weretreated with modified B16 cells containing IL-2 and GM-CSF, there was aslight prolongation of the animal's life relative to controls. No micewere cured. Conversely, treatment with the IL-2, GM-CSF, TNF-αcombination resulted in the apparent cure of 3 out of 5 mice, asevidenced by survival in excess of 120 days. Two of the three micesurvived a subsequent challenge of wild-type B16 cells. The addition ofIL-4, ICAM or CD2, individually, to IL-2 and GM-CSF expressing cellsresulted in a apparent cure of 1 out of 5 mice (the mice survived up tothe end of the study, and displayed no overt signs of tumor-associatedillness). If treatment with the modified IL-2, GM-CSF, and TNF-α cellsbegan 3 days after the establishment of the tumor, more animals wereapparently tumor free, and, in some groups, 10 out of 10 mice wereapparently cured.

Reversal of established tumors was also shown using irradiated cells, asoutlined in Example 9. As shown in FIG. 3C, irradiated B16 cellsexpressing GM-CSF were capable of mediating the rejection ofpre-established tumors, while irradiated non-transduced cells were not,at least at the doses tested. FIG. 3C shows that the extent ofprotection was dependent upon both the dose of challenge cells and thetime at which the therapy was initiated. Similar results were alsoobtained in studies in which established metastases were generatedthrough the intravenous injection of non-transduced cells (data notshown),

7.11. Characteristics of the Immune Response Elicited by GM-CSFExpressing Tumor Cells

The immune response elicited by vaccination with irradiated B16 melanomacells expressing GM-CSF was characterized in a number of ways.Histological examination of the site of injection of irradiated GM-CSFexpressing cells five to seven days after injection revealed anextensive local influx of immature, dividing monocytes, granulocytes(predominantly eosinophils), and activated lymphocytes (FIG. 5, panelA). Within one week after injection, the draining lymph mode haddramatically enlarged, showing paracortical hyperplasia and somegerminal center formation (FIG. 5, panel C). In contrast, in micevaccinated with non-transduced irradiated cells, only a mild influx ofinflammatory cells was seen (FIG. 5, panel B), which consisted primarilyof lymphocytes, and a comparatively smaller enlargement of the draininglymph node was observed (FIG. 5, panel D). At the challenge site of micevaccinated with irradiated GM-CSF expressing cells (FIG. 5, panel E), alarge number of eosinophils, monocytes, and lymphocytes were evidentafter five days, while only patches of lymphocytes were seen at thechallenge site in mice vaccinated with irradiated cells (FIG. 5, panelF). Virtually no responding cells were observed in naive animalschallenged with live B16 cells (FIG. 5, panel G).

To determine which cells were critical for systemic immunity, a seriesof mice were depleted of CD4+, CD8+, or natural killer cells by theadministration of antibodies in vivo and subsequently vaccinated withirradiated GM-CSF expressing cells. Specifically, beginning one weekprior to vaccination, mice were depleted of either CD4+ cells with MabGK1.5 CD8+ cells with Mab 2.43, or NK cells with PK 136. The antibodieswere partially purified by ammonium sulfate precipitation:delipidatedascites was mixed 1:1 with saturated ammonium sulfate and theprecipitate was collected by centrifugation, dried, and resuspended in avolume of dH₂ O equivalent to the original volume of ascites. Theantibody was dialyzed extensively against PBS and passed through a 0.45μm filter. Antibody titre was tested by staining 1×10⁶ splenocytes withserially diluted antibody and determining saturation by FACSCANanalysis. All preparations were titred beyond 1:2000. Antibodies wereinjected at a dose of 0.15 ml i.u. and 0.1 ml i.p. on day 1 ofdepletion, and 0.1 ml i.p. every week thereafter. Depletion oflymphocyte subsets was assessed on the day of vaccination (earlydepletions) on the day of wild type tumor challenge (early and latedepletions), and weekly thereafter. Flow cytometric analysis of lymphnode cells and splenocytes stained with 2.43 or GK1.5 followed byfluorescein isothiocyanate labeled goat antibody to rat IgG or with Pkl36 followed by fluorescein isothiocyanate-labeled goat antibody to mouseIgG revealed that the depleted subset represented <0.5% of the totallymphocytes, with normal levels of the other subsets present. Both CD4+and CD8+ T cells were required for effective vaccination, sincedepletion of either T cell subset prior to vaccination abrogated thedevelopment of systemic immunity, whereas depletion of NK cells hadlittle or no effect (FIG. 6, panel A).

In addition, when various T cell subsets were depleted subsequent toimmunization but prior to challenge, both CD4+ and CD8+ T cells wereagain found to be important (data not shown), thus indicating a role forboth subsets at the effector as well as the priming phase of theresponse.

Finally, the generation of tumor specific CD8+ cytotoxic T cells in micevaccinated with either non-transduced irradiated cells or irradiatedcells expressing GM-CSF was examined. In this series of experiments, 14days after vaccination with either irradiated GM-CSF transduced ornon-transduced B16 cells, splenocytes were harvested and stimulated invitro for 5 days with gamma-interferon treated B16 cells. CD8 blockableCTL activity was determined in a 4 hour ⁵¹ Cr release assay ongamma-interferon treated B16 targets at various effector:target ratios.Splenocytes from naive C57B1/6 and Balb/c mice served as controls. Thedata are shown in FIG. 6B, where the hollow square represents GM-CSF,the solid circle represents Allo, the hollow diamond represents naiveand the solid triangle represents B16 irradiation.

While mice vaccinated with non-transduced cells possessed littledetectable CD8+ blockable killing, the level of CD8 blockable killingwas significantly enhanced in sample of cells isolated from micevaccinated with GM-CSF expressing cells.

Previous studies had suggested that cytokine expressing tumor cellsmight enhance the generation of systemic anti-tumor immunity through theability of cytokine expression to bypass T cell help (8). This model wasbased on the finding that IL-2 expressing tumor cells elicited systemicimmunity in both the CT-26 colon carcinoma and B16 melanoma model, andthat the tumor immunity depended only upon CD8+ cells. However, theinability to generate systemic immunity with IL-2 expressing B16 cellsand the dependence of the GM-CSF effect upon CD4+ cells suggests thatGM-CSF expressing cells may elicit systemic immunity in a fundamentallydifferent manner.

Several characteristics of the immune response induced by vaccinationwith irradiated cells expressing GM-CSF suggest that the underlyingmechanism may involve enhanced presentation of tumor-specific antigensby host antigen presenting cells. While the extent of systemic immunityinduced by GM-CSF expressing cells was not appreciably affected by thelevel of GM-CSF secretion examined (FIG. 3B), the absolute number ofvaccinating tumor cells was critical. This finding suggests that theabsolute amount of tumor specific antigen available for vaccination maynormally be limiting. Also, the injection of GM-CSF expressing cells ledto a dramatic influx of monocytic cells, known to be potent antigenpresenting cells (57), with lesser numbers of other cell types.Furthermore, both CD4+ and CD8+ cells were necessary for the immuneresponse. Since the B16 cells used in this invention do not expressdetectable amounts of class II MHC molecules, even after γ-IFN treatment(unpublished results), it is likely that host antigen presenting cells,rather that the tumor cells themselves, were responsible for the primingof CD4+ T cells. These data are consistent with the hypothesis thatlocal production of GM-CSF modifies the presentation of tumor antigenssuch that collaboration between CD4+ helper and CD8+ cytotoxic killer Tcells is established or improved in vivo.

7.12. Use of Other Retroviral Vectors

pLJ: The characteristics of this vector have been described in (20), andin U.S. Ser. No. 07/786,015, filed Oct. 31, 1991, and in PCT/US91/08121,filed Oct. 31, 1991. This vector is capable of expressing two types ofgenes: the gene of interest and dominant selectable marker gene, such asthe neo gene. pLJ vectors containing murine IL-2, GM-CSF, IL-4, IL-5,γ-IFN, IL-6, ICAM, CD2 TNF-α, and IL1-RA (interleukin-1-receptorantagonist) are made. In addition, human sequences encoding TNF-α,GM-CSF and IL-2 are constructed. The gene of interest is cloned indirect orientation into a BamHI/SmaI/SalI cloning site just distal tothe 5' LTR. The neo gene is placed distal to an internal promoter (fromSV40) which is farther 3' than the cloning site (i.e. it is located 3'of the cloning site). Transcription from pLJ is initiated at two sites:the 5' LTR, which is responsible for the gene of interest, and theinternal SV40 promoter, which is responsible for expression of theselectable marker gene. The structure pLJ is represented in FIG. 1B.

pEm: In this simple vector, described in U.S. Ser. No. 07/786,015, filedOct. 31, 1991, abandoned, and in PCT/US91/08121, filed Oct. 31, 1991,the entire coding sequence for gag, pol, and env of the wild type virusis replaced with the gene of interest, which is the only gene expressed.The components of the pEm vector are described below. The 5' flankingsequence, 5' LTR and 400 base pairs of contiguous sequence (up to theBamHI site) are from pZIP. The 3' flanking sequence and LTR are alsofrom PZIP; however, the ClaI site 150 base pairs upstream from the 3'LTR has been ligated with synthetic BamHI linkers and forms the otherhalf of the BamHI cloning site present in the vector. The HindIII/EcoRIfragment of pBR322 forms the plasmid backbone. This vector is derivedfrom sequences cloned from a strain of Moloney Murine Leukemia virus.

pEm vectors containing murine IL-2, GM-CSF, IL-4, IL-5, γT-IFN, IL-6,ICAM, CD2 TNF-α, and IL-1-RA (interleukin-1-receptor antagonist) aremade. In addition, human sequences encoding TNF-α, GM-CSF and IL-2 areconstructed.

αSGC: The α-SGC vector, described in U.S. Ser. No. 07/786,015, filedOct. 31, 1991, abandoned and in PCT/US91/08121, filed Oct. 31, 1991,utilizes transcriptional promoter sequences from the α-globin gene toregulate expression of the cytokine-encoding gene. The 600 base pairfragment containing the promoter element additionally contains thesequences for the transcriptional initiation and 5' untranslated regionof the authentic α-globin mRNA. A 360 base pair fragment which includesthe transcriptional enhancer from cytomegalovirus precedes the α-globinpromoter and is used to enhance transcription from this element.Additionally, the MMLV enhancer is deleted from the 3' LTR. Thisdeletion is transferred to the 5' LTR upon infection and essentiallyinactivate the transcriptional activating activity of the element. Thestructure of α-SGC is represented in FIG. 1D.

α-SGC vectors containing murine IL-2, GM-CSF, IL-4, IL-5, γ-IFN, IL-6,ICAM, CD2 TNF-α, and IL-1-RA (interleukin-1-receptor antagonist) aremade. In addition, human sequences encoding TNF-α, GM-CSF and IL-2 areconstructed.

7.13. Co-administration of Viral Antigens and Cytokines

An appropriate vaccinating cell (which may include but not be restrictedto, fibroblasts, keratinocytes, endothelial cells, monocytes, dentriticcells, B-cells or T-cells) would be infected with a retrovirusexpressing an individual or combination of HIV antigens (including, butnot limited to gag, env, Dol, rev, nef, tat, and vif), and a single orcombination of cytokines (such as GM-CSF), irradiated or otherwiserendered proliferation incompetent, and administered to a patient.

7.14. Suppression of Immune Responses

An appropriate vaccinating cell (see example 13) as well as particularcells of an allograft (i.e. renal tubular epithelial cells in thekidney) would be infected with a virus expressing a single orcombination of cytokines (such as gamma-interferon), irradiated orotherwise rendered proliferation incompetent, and administered to apatient to reduce the immune response against the allograft.

Additionally, the present invention can be used to suppress autoimmunediseases. The autoimmune response against a host target antigen couldalso be modified in a similar way. For example, the present inventioncould be used to treat multiple sclerosis. An appropriate cell (seeexample 13) would be infected with a retrovirus expressing myelin basicprotein as well as a cytokine (such as gamma-interferon) to reduce theimmune response directed against myelin basic protein in the patient.

7.15. Enhancement of Immune Response

It is also possible to use the present invention to enhance the immuneresponse to a variety of infectious diseases. For example, the presentinvention could be utilized in the treatment of malaria. An appropriatevaccinating cell (see example 13) would be infected with a retrovirusexpressing the gene form circumsporouzoite surface protein and acytokine (such as GM-CSF), irradiated or otherwise renderedproliferation incompetent, and them administered to a patient to induceprotection of a therapeutic response against malaria.

7.16. Human Clinical Studies 7.16.1. Melanoma Trials

Melanoma patients in which the disease had progressed to a stage wherethe surgery did not remain as an effective option were used in thestudy. The study also required that the patients show no evidence ofautoimmune disease, serious allergy, or brain metastases (as determinedby magnetic reasonance imaging, MRI), and retained adequate immune,hepatic, renal, and bone marrow function.

The huGM-CSF gene was subcloned into the insertion site of theretroviral vector MFG-S (a derivative of MFG) in a proper context andorientation for expression of the gene. The recombinant vector wassubsequently transfected into the amphotropic packaging cell line pCRIPand high producer transfectants were isolated. High titer stocks ofhelper free retroviral particles which harbor the huGM-CSF gene werethen harvested from the culture medium of the transfected packaging celllines essentially as described above.

Tumor cells were resected from cutaneous and subcutaneous lesions, lymphnodes, and lung, liver, and soft tissue metastases. After resection, thespecimens were aseptically transferred to a sterile container andtransported to the laboratory. All subsequent procedures were conductedunder aseptic conditions. The tumor tissues were disaggregated by eithercollagenase treatment or by mechanical dissociation and subsequentlygrown in culture and/or frozen in DMSO with 50 percent FCS (collagenasetreated) or human albumin (mechanical dissociation). The numbers of theprimary and secondary autologous tumor cells were expanded in culture,and a portion of the cells were subsequently transduced to producehuGM-CSF using an MFG-S construct.

After verification that the transduced cells were sterile, viable andproducing cytokine, (see Table 2), a total of either 5×10⁶ (low dose) or5×10⁷ (high dose) of the transduced tumor cells were irradiated,suspended in normal saline, and injected (both intradermally andsubdermally at up to five different sites in volumes of 0.25-1.0 ml)back into the original donors. Up to three injections at the givendosages were applied to the individuals at approximately 21 dayintervals. Control individuals were seperatively injected withirradiated non-transduced cells and levels of GM-CSF analogous to theamounts of GM-CSF produced by the transformed cells which were injectedinto the test subjects.

                  TABLE 2    ______________________________________    GM-CSF Production (ng/10.sup.6  cells/24 hrs)    TEST SUBJECT  GM-CSF PRODUCTION    ______________________________________    (Low Dose)    001           168.2    002           251.6    006           124.6    008            97.1    010            46.0    011           123.6    020           180.0    (High Dose)    018           187.3    019           128.3    ______________________________________

After injection, tests were performed to confirm that the transdcuedtumor cells elicited an immune response. These tests included assayingfor delayed type hypersensitivity (DTH) reactions to intradermallyinjected (irradiated) tumor cells, and biopsies of the vaccination andinjection sites. The data indicate that T-cells are present in theinflamed tissues, and that there is a reporducible increase in thenumber of monocytes. The control subjects (who were injected with eithernontransduced tumor cells or huGM-CSF at levels analogous to thoseproduced by the transduced antologus tumor cells) showed little to noneof the differences seen in the test subjects. Immunohistochemistryindicated that a tendency for antigenic drift correlated with decreasedexpression of the Mel 4 and HMB45 markers.

Additional tests were conducted to determine whether or not an increasein the number of eosinophils correlated with the injections. As seen inFIG. 10, after each vaccination, a transient increase in the percentageof eosinophils in the bloodstream generally followed.

The above data indicate that immune stimulation had occurred, and thatthe observed immune stimulation could be associated with the fact thatthe autologous tumor cells were expressing huGM-CSF in vivo.

7.16.2. Renal Cell Carcinoma Trials

Eighteen patients with advanced renal cell carcinoma were vaccinated upto four times (at approximately one month intervals) with either 4×10⁶,4×10⁷, or 4×10⁸ transduced and irradiated tumor cells. Tumor tissue wassurgically resected (see above), and the population of primary andsecondary tumor cells was expanded by in vitro culture. The culturedcells were subsequently transduced with the huGM-CSF/MFG-S constructdescribed above, and tested for GM-CSF production. Transduced autologouscells which produced acceptable quantities of cytokine were irradiatedand injected into the donor individuals as described above.

At the lowest dose of injected cells, the levels of GM-CSF secretedranged between about 17 and 189 ng/10⁶ cells/24 hrs. At the middle dose,the transduced cells constructed from the various individuals' tumorssecreted between about 42 and 149 ng of GM-CSF/10⁶ cells/24 hrs.

Control individuals were injected with irradiated nontransduced tumorcells. At the lowest dose, three subjects received non-transduced cells,at the middle dose of cells, five subjects received non-transducedcells, and the two patients that were injected with the highest dosageof cells only received nontransduced cells.

Patient responses to the vaccinations included marked erythema andinduration at the injection site (Dose 1 showed the least response),which became more pronounced after multiple vaccinations (this wasespecially the case when transduced cells were injected).

Moreover, test subjects who were multiply injected with transduced cellsalso displayed positive DTH reactions, as well as an increase in theamount of eosiniphils (following vaccination). As in the melanoma study,apart from local symptoms (erythema, induration and itching), nosignificant adverse events were observed.

The above data reveal that the injection of irradiated autologous tumorcells expressing GM-CSF into human test subjects stimulated anobservable in vivo tumor-specific immune response against the parenttumor cells.

It will be understood by those skilled in the art that variousmodifications of the present invention as described in the foregoingexamples may be employed without departing from the scope of theinvention. Many variations and modifications thereof will be apparent tothose skilled in the art and can be made without departing from thespirit and scope of the invention herein described.

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EOUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

We claim:
 1. A method of stimulating a systemic immune response of ahuman to an established tumor, comprising administering to the human atherapeutically effective amount of tumor cells, wherein said tumorcells have been rendered proliferation incompetent by irradiation andhave been genetically engineered to express granulocyte-macrophagecolony stimulating factor, and wherein said tumor cells elicit asystemic immune response to said established tumor, and wherein saidestablished tumor and said tumor cells are of the same type.
 2. A methodof stimulating a systemic immune response of a mammal to an establishedtumor comprising: administering tumor cells, wherein said tumor cellshave been rendered proliferation incompetent by irradiation and havebeen genetically engineered to express granulocyte-macrophage colonystimulating factor, and wherein said tumor cells elicit a systemicimmune response to said established tumor, and wherein said establishedtumor and said tumor cells are of the same type.
 3. The method of claim2 wherein said tumor cells further have been genetically engineered toexpress a protein selected from the group consisting of: IL-4, IL-6, CD2and ICAM.
 4. A method of suppressing growth of an established tumor in amammal, comprising administering to said mammal: tumor cells whereinsaid tumor cells have been rendered proliferation incompetent byirradiation and have been genetically engineered to expressgranulocyte-macrophage colony stimulating factor and wherein said tumorcells elicit a systemic immune response to said established tumor suchthat growth of said established tumor is suppressed, and wherein saidestablished or an said tumor cell are of the same type.
 5. The method ofany one of claims 2, 3, or 4, wherein said mammal is a human.
 6. Themethod according to claim 5 wherein said granulocyte-macrophage colonystimulating factor is of human origin.
 7. A method of suppressing growthof melanoma in a human comprising the step of administering to saidhuman a therapeutically effective amount of proliferation-incompetentmelanoma cells that have been genetically engineered to expressgranuloyte-macrophage colony stimulating factor, wherein said melanomacells elicit a systemic immune response to said melanoma.
 8. The methodof claim 7, wherein said proliferation-incompetent melanoma cells areirradiated.
 9. A method of suppressing growth of melanoma in a mammalcomprising the step of administering to said mammal a therapeuticallyeffective amount of proliferation-incompetent melanoma cells that havebeen genetically engineered to express granulocyte-macrophage colonystimulating factor, wherein said melanoma cells have been renderedproliferation incompetent by irradiation and wherein said melanoma cellselicit a systemic immune response to said melanoma.